In this proposal, we plan to investigate the energy transducing ATPase (CF0-CF1) complex associated with the thylakoid membranes of higher plants and the green algae Chlamydomonas reinhardi. Part I: Mechanistic problems related to the higher plant CF0-CF1 complex. Section A: Photoaffinity labels (2-azido adenine nucleotides) will be used to locate and identify the nucleotide regulatory binding site on membrane-bound CF1. Section B: (i) Distereomers of nucleoside phosphothioates will be used to determine the absolute stereochemistry of the ATPase reaction catalyzed by soluble CF1. (ii) Isotope trapping experiments will be performed to determine the kinetic mechanism of ATP synthesis and the binding constant for the first substrate to the ATP synthase active site. Section C: We will investigate the reversibility of the membrane-bound CF1 ATPase. Part A: We will investigate the mechanism of the reversible solvent-induced activation of the soluble ATPase using both physical and chemical methods. Part B: We will isolate and purify the CF0-CF1 complex from C. reinhardi. Part C: We plan to investigate the biochemical basis for the inability of isolated thylakoid membranes from C. reinhardi strain 2137 to catalyze photophosphorylation when cultured in the dark. Part D: We will measure the turnover of membrane-bound CF1 and the pool sizes of its subunits. SECTION D: We plan to isolate mutants of C. reinhardi defective in CF1 and characterize the resultant polypeptides.